Tendon development is the result of multiple regulatory pathways converging to produce and maintain a structure that results in a functional tendon. The overall goal of this application is to define the mechanisms that regulate the sequential events in tendon extracellular matrix assembly. Defining the regulatory pathways essential for normal development, growth and maturation will provide the basis for understanding tendon development. In addition, tendons have a limited capacity for regeneration and developmental paradigms may be applied to promote regenerative healing as well as in the creation of treatment modalities that shift fibrotic repair toward regeneration and restoration of full biomechanical function. Also, this work will provide milestones needed to develop stem cell therapies and evaluate bioengineered scaffolds. Finally, the underlying molecular pathophysiology of connective tissue disorders, e.g., classic Ehlers Danlos Syndrome, a newly defined collagen XII related myopathy, is addressed. This provides a foundation for future translational studies. Our general hypothesis is that tendon matrix assembly is a hierarchal process requiring specific regulatory interactions at each level. This application addresses three key regulatory steps in the development of tendon structure and function: (1) fibril nucleation at the tenocyte surface; (2) stabilization of immature protofibrilsand assembly into fibers; and (3) fibril growth generating mature fibrils. The following hypotheses wil be addressed: (1) Collagens V/XI isoforms regulate tendon fibril assembly involving interactions with collagen I at the tenocyte surface that control the number of protofibrils nucleated, their initial diameter, and influences organization of the developing matrix. (2) Protofibril organizatio and assembly into fibers is mediated through collagens XII and XIV involving coordinate interactions of collagen XIV and collagen XII isoforms. (3) Small leucine-rich proteoglycans (SLRPs) regulate fibril growth during tendon development through synergistic interactions across classes: decorin and biglycan with lumican and fibromodulin. The specific aims of this application are to: (Aim 1) elucidate the mechanisms involving collagen V/XI isoforms in the regulation of protofibril nucleation and initial assembly; (Aim 2) define the coordinate regulatory roles of FACIT collagens XII and XIV in organization of protofibrils during deposition into fibers and fiber assembly; and (Aim 3) elucidate the mechanisms involving small leucine-rich proteoglycans (SLRPs) in the coordinate regulation of tendon fibril growth and maturation. Existing and newly created mouse models null for a specific matrix molecules will be utilized to analyze the regulation of tendon development using biochemical, immuno-chemical, molecular, morphological and functional approaches. Definition of the regulatory steps in tendon-specific matrix assembly provides a developmental paradigm for understanding of tendon repair versus regeneration, pathobiology, and the manipulation of these processes. PUBLIC HEALTH RELEVANCE: In sports, at work, or due to aging processes, tendon injuries cause significant pain and disability, resulting in enormous healthcare costs, loss of work, and a decrease in the quality of life. The focus of this application is to elucidate the mechanisms regulating tendon extracellular matrix assembly during development, growth and maturation. Tendons have a limited capacity for regeneration and developmental paradigms may be applied to promote regenerative healing and in the creation of treatment modalities that shift fibrotic repair toward regeneration and restoration of full function. Defining the mechanisms required for intercalated growth of fibrils and fibers will provide insight into repair/reintegration at bone-tendon and myotendonous interfaces providing a foundation for design of new therapeutic interventions. In addition, our data will provide milestones needed to develop stem cell therapies and evaluate bioengineered scaffolds. Furthermore, our work provides a foundation for understanding the functional defects observed in musculoskeletal diseases.